Fluorescence systems are often employed to analyze or image biological samples. In such systems, the sample is typically exposed to laser light at a wavelength at which a material of interest in the sample, such as a fluorophore or a naturally occurring substance in the material, absorbs light causing it to fluoresce or emit light at a different (typically longer) wavelength. Light emitted from the sample is then detected so that the location, amount, and other properties associated with the material of interest, as well the sample, can be determined. In addition, an image of the sample can be constructed based on the detected fluorophore, for example.
In many fluorescence systems, a single photon of laser light excites an atom in the material of interest. The atom then relaxes to a lower energy state, and, in doing so, a single photon is emitted. In so-called “multiphoton” fluorescence systems, however, multiple laser light photons simultaneously excite the atom of the material of interest into a higher energy state. When the atom relaxes, it emits a photon typically having more energy (shorter wavelength) than the energy associated with the laser photons having longer wavelengths. For example, fluorescein, a known fluorophore, strongly absorbs light at 500 nm, but can also be excited via a two-photon process at about 1000 nm. Similarly, certain naturally occurring fluorescent molecules strongly absorb light at about 350 nm, and can be excited via a three-photon process at about 1050 nm. U.S. Pat. Nos. 5,034,613, 6,166,385, and 6,344,653 describe microscope systems for measuring multiphoton fluorescence, and are incorporated herein as references.
Typically, the laser light in a multiphoton fluorescence system is applied at relatively high intensity to a localized region or focused spot in the sample. The three-dimensional focal spot of the laser light can penetrate deep into the sample, especially if longer wavelengths in the infrared are employed. Accordingly, by changing the depth of penetration and detecting a two dimensional section of the sample at each depth, a three dimensional composite image of the sample can be obtained by stacking the two dimensional sections with known imaging software. Moreover, since only the localized region emits light, only that region is imaged.
Multiphoton fluorescence occurs with a reasonably high probability when two or more laser light photons are absorbed simultaneously (thus supplying the same energy as a single photon at the main absorption wavelength). Accordingly, multiphoton fluorescence often requires high laser light intensities, or a large density of photons in a relatively short period of time. Therefore, conventional high-power pulsed lasers are often used, such as tunable Ti:Sapphire lasers, which can output light at wavelengths as short as about 680 nm and as long as about 1100 nm. With higher laser light intensities, each two dimensional section can be finely resolved and the resulting three dimensional image can be made sharper.
Relative to alternative fluorescence imaging techniques, multiphoton imaging advantageously makes it possible to: study dynamic processes in thick living cells; eliminate undesirable noise fluorescence above and below each two dimensional section; reduce undesirable photobleaching of the sample outside each two dimensional section, thereby preventing overexposure of the sample; eliminate the optical loss associated with a detector pinhole as required in other three-dimensional fluorescent imaging systems, such as confocal microscopy systems.
In addition to the lasers described above, multiphoton fluorescence systems typically include a photodetector in order to sense the emitted light. In order to reduce the amount of other light reaching detector, such as light from the laser, filters must be employed which are transmissive at wavelengths of light emitted by the sample, but reflective and/or absorbing at other wavelengths. If light at such other wavelengths is adequately suppressed, a so called “spectral darkfield” can be achieved in which an image is black or dark when no features of interest are present. Image quality can thus be improved. Without this spectral darkfield property, in most samples no fluorescence could be observed.
Optical filters are also used to direct the laser light to the sample, and if highly reflective or transmissive at wavelengths associated with the emitted light, can efficiently direct the emitted light toward the photodetector.
With improved optical filters, more photons of emitted light and fewer photons of undesired light (e.g., the laser light) are fed to the photodetector. Thus, weaker signals can be detected, or less laser light is required to generate a given emitted optical signal, thereby minimizing damage to the sample by intense laser light. Or, an image can be detected in less time leading to higher speed measurements. A higher signal-to-noise ratio (and therefore better resolution) can be achieved in the image, since, for example, the filter can block more laser light from reaching the photodetector, while transmitting a given intensity of emitted light. Also, systems having additional functionality, such as a systems that can accurately sense light over a broader range of emission wavelengths, can be achieved. Such systems can include filters having relatively wide transmission wavelength bands, as well as wide blocking wavelength bands. As a result, new fluorophores or fluorescent biological substances can be excited efficiently, thereby improving image quality and increasing the number of different types of samples that can be imaged.
Different fluorophores emit light at different wavelengths. In addition, the laser in a multiphoton fluorescence system should output light over a broad range of wavelengths in order to excite a wide range of fluorophores. Thus, in order to be used in connection with most known fluorophores, a filter in a multiphoton fluorescence system typically should have high transmission over a wide range of wavelengths. In addition, the filter should have high blocking over the entire range of laser wavelengths, e.g., those wavelengths associated with a Ti:Sapphire laser. Typically, however, it is difficult to achieve both high blocking and high transmission over such wide wavelength ranges. Conventional filters, therefore, either have narrower blocking or transmission bands, or the level of transmission and/or blocking is less than optimum. For example, known filters provide transmission in excess of 50% only in a range of about 410 nm to 680 nm, and reach 90% in a narrow range within this band. In addition, such filters may have an optical density (“OD” where OD=−log10(T), T being transmission of the filter at a particular wavelength) less than 6 at least at certain Ti:Sapphire wavelengths, which may be inadequate when such wavelengths are required. Higher OD values can be achieved, but at the expense of creating second- and third-order stop bands of reduced transmission at selected emission wavelengths.
These higher-order “stop bands” are one reason why it is difficult to achieve high transmission at wavelengths shorter than those over which high blocking occurs. A stop band is a range of wavelengths over which transmitted light is strongly attenuated due to constructive interference of the many partial waves of light reflected off of a structure with a periodic or nearly periodic variation of the index of refraction, as found in a thin-film interference filter. For a “quarter wavelength stack” structure comprised of alternating layers of high- and low-index materials, each of which is approximately one quarter of a particular wavelength λ0 thick (in the material), the “fundamental” stop band is roughly centered on λ0 and ranges from approximately λ0/(1+x) to λ0/(1−x), where x is related to the high and low index of refraction values, nH and nL, respectively, according to
  x  =            2      π        ⁢                  arcsin        ⁡                  (                                                    n                H                            -                              n                L                                                                    n                H                            +                              n                L                                              )                    .      If the layer-to-layer index of refraction variation is not a purely sinusoidal variation, but rather changes abruptly, as is typically the case in a multi-layer thin-film interference filter, higher-order stop bands exist at shorter wavelengths. For example, a quarter-wave stack having such abrupt refractive index changes exhibits “odd-harmonic” stop bands that occur approximately at the wavelengths λ0/3, λ0/5, etc., and where these stop bands range from approximately λ0/(3+x) to λ0/(3−x), for the third-order stop band, λ0/(5+x) to λ0/(5−x), for the fifth-order stop band, and so on. If the layers are not exactly a quarter-wave thick, then there may also be “even-harmonic” stop bands that occur approximately at the wavelengths λ0/2, λ0/4, etc. In general, high blocking over a wide range is achieved by utilizing a fundamental stop band, by combining multiple fundamental stop bands, or by “chirping” (gradually varying) the layers associated with one or more fundamental stop bands. Regardless of the approach, the higher-order harmonic stop bands associated with these blocking layers inhibit transmission at wavelengths shorter than the fundamental stop band or stop bands.
Other filters are known which have a relatively wide transmission band, but the transmission within the band is reduced. Still other filters can achieve moderately high transmission within a range of about 430 nm to 710 nm and moderately high reflection or blocking from about 780 nm to 1100 nm. The transmission characteristic of these filters, however, has a relatively shallow slope for wavelengths between the transmission and the blocking bands. As a result, light at such wavelengths may be detected by the photodetector, thereby reducing image quality.